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Epistasis
The purpose of this page is to act as a tutorial for the epistasis experiment. If this is your first time doing any of the protocols listed in this page, I would advise you to be under the supervision of the yeast people in the lab. I AM ADDING SOME STUFF THAT MARK LEFT OUT This experiment consists of protocols that already exist on the wiki, so I will be referencing them most of the time. This experiment is set up to be a two-week experiment, starting from pulling strains leading up to the dissection of tetrads. Then another week or so to colony PCR and run a gel on the desired tetrads to verify the deletions. Now that that is out of the way, we can begin. This is rough estimate of the double deletion strains that need to be made. If you need to figure out which strains are done/undone, go to Marc Ting because he has record of all of it. From here, you need to figure out 25 strains that still need to be made (note: make sure you don’t make homozygous diploid strains). From here, you will need to determine which strains you need to pull strains from the deletion collection by looking at the CORE database. If you do not yet have a login, then ask either Mark or Scott for one. It's OK to pull strains for just one mating type for each deletion, but for safety, I usually pull both mating types for deletion. For example, if I am trying to pull gpa2 strain, I would pull both MATa and MATalpha. The reason I do this is if for some reason, one of them don't grow, you can still mate it using the mating type that did grow, just make sure you make a note of it. If you followed the table above and pulled the strains on Monday, then wait two days for them to fully grow. By Wednesday, you should be able to mate the strains. Follow this protocol to do just that. Wait for two days again. By Friday, the colony should now be big enough for you to play around with. Following the table, you should start an overnight of diploid strains you now made, so that you can freeze them the next day. In addition to this, we could start sporulating the diploids. Make sure that you have made enough plates for all of the strains. When I'm labeling the plates, one trick I do is duplicate the pies to the second GNA patch that way I can simply replicate these the next day. The next day (Saturday), all you would have to do is patch the strains onto another second GNA patch. Again, you could just do this by replicating them. Then on Sunday, they can be placed on to SLSM media, following the GNA sporulation protocol, and placed in the tube roller. From here on out, you wait 5 days until they can be switched to the 30C shaker. Then wait another 3 days. Two weeks from when you started, you are now able to dissect tetrads. I typically do 20 tetrads just to be safe. My ongoing rate without blowing my brains off is 5 plates a week, though this may increase as time passes. Tetrads take about two days to grow (three if you want to make sure you have large enough colonies). From here they need to be genotyped. This step can be done plenty of ways, but the way I do is replica plate them onto a total of 8 plates: G418, -Ura, -Met, -Lys, and two mating tester plates in this same order. The mating tester plate is made by grabbing a toothpick full of the correct query strains, and placing it into 1 mL of ddH2O and vortexing. Then, they need to be spread throughout two separate YPD plates. The way I replicate these guys is get the YPD and replicate them on the velvet, then placing each of the dropout plates on the velvet. I only switch velvets on the last plate, because you don't want to replicate onto the plate that has a different mating tester. Again, if this is your first time, this step will probably need to be showed to you. The next day, the mating tester strains should be replicated on basic plates. Then wait for one more day. Now, we can score all of the tetrads. Using the tetrad score sheet , you can determine which tetrad you could possibly colony PCR. The point of doing this step is to determine whether or not the strain you have is legitimate or not. The protocol for colony PCR'ing is here. Once you have determined the spore you want, you can start an overnight of it and freeze it the next day. Remember to label it with the correct strain name.